Synthesis of des-Ala{hu 1{b , Gly{hu 2{b , Asn{hu 5{b -SRIF and intermediates

ABSTRACT

The undecapeptide H-Cys-Lys-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-CysOH, its oxidized form and intermediates obtained in such synthesis are described. The oxidized form of this undecapeptide inhibits the secretion of the hormone somatotropin.

United States Patent n91 Sarantakis [4 1 May 6,1975

[ SYNTHESIS OF DES-ALA, GLY ASN -SRlF AND INTERMEDIATES [75] Inventor:

[73] Assignee: American Home Products Corporation, New York. NY.

[22] Filed: Jan. 3, 1974 [21] Appl. No.: 430,441

Dimitrios Sarantakis, Audubon, Pa.

[52] US. Cl. 260/1125; 424/[77 [51] Int. Cl C07c 103/52; C07g 7/00 [58]Field of Search 260/1125 [56] References Cited OTHER PUBLICATIONSBrazeau et al.: Science, I79, 77-9 (1973).

G. R. Pettit, Synthetic Peptides," Vol. 1. Van Nostand, Reinhold Co.,New York (1970), pp. [UL 464.

Primary ExaminerElbert L. Roberts Assistant ExaminerReginald .I. Suyat(57] ABSTRACT 6 Claims, N0 Drawings r 1 SYNTHESIS OF DES-ALA, GLY, ASN-SRIF AND INTERMEDIATES This invention relates to novel undecapeptidesand intermediates used in their synthesis by the classical method ofpeptide synthesis.

Somatostatin (also known as somatotropin release inhibiting factor-SRIF)is the tetradecapeptide.

l-l-Ala-Gly s-L s-Asn-Ph -Phe-Trp-Lys-Thr-Phe- Thr-Ser-Cys-OH.

This tetradecapeptide has only recently been identified by isolationfrom extracts of ovine hypothalamic tissues and found to inhibit thesecretion of the hormone st matotropin which is commonly referred to asthe growth honnone (GI-l); See Brazeau et al., Science, 179 pp 77-79(January 1973). The linear form of this tetradecapeptideH-Ala-Gly-Cys-Lys-Asn-Phe-Phe- Trp-Lys-Thr-Phe-Thr-Ser-Cys-OH, has alsobeen reported by Brazeau et al., supra, to have been synthesized bysolid phase methodology and found to have the same biological activityas the somatostatin obtained from a natural source.

The novel undecapeptides of the present invention are analogs ofsomatostatin and the linear counterpart of somatostatin in which theamino acids in the one, two and five position of somatostatin have beenomitted.

The nomenclature used to depict the peptides follow that shown is bySchroder & Lubke, The Peptides," 1 pp viii-xxix (Academic Press l965)and in accordance with such nomenclature, it is the L form of the aminoacid that is intended, unless otherwise expressly indicated.

The undecapeptide of the present invention which inhibits the secretionof the hormone somatotropin is represented by the formula:

H-Cys-Lys- Phe- Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys-OH and the non-toxicacid addition salts of such compounds.

lllustrative of acid addition salts are hydrochloride, hydrobromide,sulfate, phosphate, maleate, acetate, citrate, benzoate, succinate,malate, ascorbate, and the like.

The present invention also relates to novel undecapeptide intermediatesof the formula:

H-Cys-Lys-Phe-PheJrp-Lys-Thr-Phe-Thr-Ser-Cys-OH and groups illustratedby benzyloxycarbonyl and substituted benzyloxycarbonyl such asp-chlorobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl,p-bromobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl; (3) aliphaticurethan protecting groups illustrated by tertbutyloxycarbonyl,diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl,allyloxycarbonyl; (4) cycloalky] urethan type protecting groupsillustrated by cyclopentyloxycarbonyl, adamantyloxycarbonyl,cyclohexyloxycarbonyl; (5) thio urethan type protecting groups such asphenylthiocarbonyl; (6) alkyl type protecting groups as illustrated bytriphenylmethyl (trityl), benzyl; (7) trialkylsilane groups such astrimethylsilane. The preferred a-amino protecting group defined by R istert-butyloxycarbonyl;

R is a protecting group for the sult'hydryl group on the cysteinyl aminoacid residue in the undecapeptide. Illustrative of R is a group selectedfrom the class consisting of benzyl; substituted benzyl wherein thesubstituent is at least one of methyl, methoxy, nitro (e.g.pmethylbenzyl, p-nitrobenzyl, 2,4,6-trimethylbenzyl, etc.,); trityl,benzyloxycarbonyl, benzhydryl, p methoxybenzyloxycarbonyl,benzylthiomethyl, ethylcarbonyl, thioethyl, tetrahydropyranyl,acetamidomethyl, benzoyl, sulfate salt, etc.

R is a protecting group for the side chain amino sub- 1 stituent oflysine or R is hydrogen which means there is no protecting group on theside chain amino substituent. Illustrative of suitable side chain aminoprotecting groups are benzyl, chlorobenzyloxycarbonyl,benzyloxycarbonyl, tosyl, 2,4 dinitrophenyl, t-amyloxycarbonyl,t-butyloxycarbonyl, etc. The selection of such a side chain aminoprotecting group is not critical except that it must be one which is notremoved during cleavage of the a-amino protecting group during thesynthesis until the peptide of the desired amino acid sequence isobtained. Hence, the a-amino protecting and side chain amino protectinggroup cannot be the same;

R and R are protecting groups for the alcoholic hydroxyl group ofthreonine and serine and is selected from the class consisting ofacetyl, tosyl, benzoyl, tertbutyl, trityl, benzyl and benzyloxycarbonyl.The preferred protecting group is benzyl; or R and R is hydrogen whichmeans there is no protecting group on the alcoholic hydroxyl function;the selection of these protecting groups is not critical except that itmust be one which is not removed during cleavage of the a-aminoprotecting group during the synthesis until the peptide of the desiredamino acid sequence is obtained;

R is a-carboxyl protecting group which is stable under the processconditions used to remove the a-arnino protecting group until thepeptide of the desired chain length has been formed. illustrative of Ris a group selected from the class consisting of C C, alkyl (e.g.methyl, ethyl, butyl, pentyl, isobutyl, etc.,); benzyl; substitutedbenzyl wherein the substituent is selected from at least one of nitro,methoxy and methyl (e.g. p-methoxybenzyl, p-nitrobenzyl, 2,4-dimethoxybenzyl, 2,4,6-trimethylbenzyl, pentamethylbenzyl), phenacyl,phthalimidomethyl, B-methylthioethyl, 4-picolyl and4-(methylthio)phenyl. Preferably R is C,-C, alkyl, benzyl or substitutedbenzyl.

Also contemplated within the scope of the present invention aredipeptide intermediates of the formula:

wherein:

R and R have the same meaning as previously defined in connection withformula III; R is an a-amino protecting group as previously defined; and

R is either hydrogen or is selected from a member defined by R", supra.

The undecapeptides of formulas I through Ill are prepared in accordancewith the reaction scheme shown in the flow diagram appended hereto. Withreference to such flow diagram the compound of Formula A, R- Thr(R)-Ser(R)-OH is reacted with a carboxyl group activating reagent to forma carboxyl group activated derivative of the compound of Formula A whichis then coupled with a carboxylic acid ester of cysteine (Formula B) ata temperature between about 30 and +30C to form acompound of Formula C.The compound of Formula B, which is preferably in the form of a salt,may be present in the reaction medium while the carboxyl group activatedderivative of a compound of Formula (A) is being formed or it may beadded to the reaction vessel after the activated compound has beenformed; The coupling is carried out throughout the synthesis in thepresence of a inert organic solvent such as dichloromethane,acetonitrile, dimethylformamide, chloroform, dioxane, toluene, methylenechloride, etc. if the compound of Formula 8 is added to the reactionmedium as an acid addition salt, an acid acceptor is included inthe'reaction medium so that a free base is formed in situ which reactswith the activated derivative of a compound of Formula A. Suitable acidaccep tors include tertiary amines (e.g. triethylamine, pyridinje,quinoline, dimethylaniline, etc.,) alkali metal carbonates or other acidbinding agents known in the art.

Following the formation of a compound of formula (C), the a-aminoprotecting group on threonine is then cleaved to yield the tripeptide offormula (D). The cleaving reagent is one which will remove the oz-aminoprotecting group without cleavage of the side,chain protecting groups. Aparticular suitable reagent is trifluoroacetic acid which will cleavetertbutyloxycarbonyl off the a-amino group but not benzyl or substitutedbenzyl side chain protecting groups. Other standard cleaving reagentsare described by Schroder & Lubke, supra, pp 72-74.

The tripeptide of formula (D) is then coupled with the dipe'ptide offormula (E) after the free carboxyl group of the latter has beenactivated as previously described, this reaction being carried out inthe same manner as described in connection with the formation of thetripeptide of formula (C). Following the forma tion of the pentapeptideof formula (F), the a-amino protecting group is cleaved off threonineusing trifluoroacetic acid or other suitable cleaving reagent to form acompound of formula (G), which may be in the form of a salt depending onthe nature of the cleaving rea gent used. This pentapeptide is coupledwith a carboxyl group activated derivative of a compound of formula (H)to obtain the nonapeptide of formula (1) which is then treated with asuitable cleaving reagent such as trifluoroacetic acid to form a salt ofa compound of formula (K). This nonapeptide is then coupled with adipeptide of formula (L) which has its carboxyl group activated with acarboxyl group activating reagent to form the undecapeptide of formula(M). Thereafter, the side chain protecting groups R,R ,R and R and thea-amino protecting group are cleaved and the ester is converted to thecorresponding acid as represented by formula (II). The cleavage oftheside chain protecting groups, a-amino protecting group and formationof the free acid may be accomplished in a single step or may beperformed step-wise depending on the selection of the cleaving reagent.A particular suitable reagent is liquid hydrogen fluoride. lfdesiredtrifluoroacetic' acid can be used to remove the a-aminoprotecting group followed by treatment with sodium in liquid ammonia.

As previously indicated, the activating reagents used in theaforedescribed synthesis are those well known in the peptide art.lllustrative of these are:-(i'l). carbodiimides (e.g.N,N-dicyclohexycarbodiimide, Nethyl N- (7-dimethylamino propylcarbodiimide); (2) .cyanamides (e.g. N,N-dibenzylcyanamide; (:3)keteimines; (4) isoxazolium salts (e.g. N-ethyl-fi-phenylisoxazolium-T-sulfonate', (5) monocyclic iiitroigen containingheterocyclic amides of aromatic character containing one through fournitrogens in the ,rin'gr-such as imidazolides, pyrazolides,1,2,4-triazolides. Specific heterocyclic amides that are useful include-N,N.'- carbonyl diimidazole N,N-carbonyl-di-l,2,4 triazole; (6)alkoxylated acetylene (e.g. ethoxyacetylene);(7) reagents which form amixed anhydride with the carboxyl moiety of the amino acid (egthylchloroformate, isobutylchloroformate) and (8) nitrogen containingheterocyclic compounds having a hydroxy group on one ring nitrogen (e.g.N- hydroxyphthalimide, N-hydroxysuccinimide, lhydroxybenzotriazole).Other activating reagents and their use in peptide coupling aredescribed by Schroder & Lubke supra, in Chapter Ill and by Kapoor', J:Pharm. Sci., 59, pp l-27, (1970). i

A particular suitable activating system for a compound of formula (A) isthe use of the combination of N,N -dicyclohexylcarbodiimide (DCC) and Nhydroxybenzotriazole which minimizes racemization. In subsequentactivating and coupling reactions, the combination of DCC andN-hydroxysuccinimide is preferred.

In selecting a particular side chain protecting group to be used in thesynthesis of the peptides of formula (I), the following rules should befollowing: (a) the protecting group must be stable to the reagent andunder the reaction conditions selected for removing the a-aminoprotecting group at each step of the synthesis, (b) the protecting groupmust retain its protecting properties (ie not be split off undercoupling conditions), and (c) the side chain protecting group must beremovable upon the completion of the synthesis containing the desiredamino acid sequence under reaction conditions that will not alter thepeptide chain.

The compound of formula (I) is obtained by air oxidation of a compoundof formula (ll). This reaction is preferably carried out by a surfaceoxidation as described in Example l9.

The following examples are illustrative of the preparation of theundecapeptides of the present invention.

EXAMPLE l N-tert-Butyloxycarbonyl-S-p-Methoxybenzy-l-L- Cysteine-N-Benzyloxy-carbonyLL-Lysine Methyl Ester then a solution of N-benzyloxycarbonyl-L-lysine methyl ester hydrochloride salt (33 gr., ca0.1 mole) and N-methylmorpholine (11 ml) in dimethylformamide (200 ml)is added and allowed to reach room temperature overnight. TheN-methylmorpholine hydrochloride salt which separates is filtered offand the filtrate evaporated to dryness. The residue is partitionedbetween ethyl acetate and water and the organic phase was washed with 5percent citric acid, water, 5 percent Na CO water, dried over Na SO andevaporated to dryness. The residue is crystallized from EtoAc-EtO-Hexane to afford a crystalline mass including solvent. The compoundcan be recrystallized from EtOAc-EQO or methanol. The yield of theabovetitled product is 53 gr (86 percent).

R, (CHCl -Methano1 25:1) 0.65; Methanol, :1) 0.85.

EXAMPLE 2 N-tert-Butyloxycarbonyl-S-p-methoxybenzyl-L- Cysteinyl-N-benzyloxycarbonyl-L-Lysine BOC-Cys(SMBzl)-Lys(Cbz)OMe (14.2 gr, 23 mmoles) from Example 1 is dissolved in methanol (ca. 100 ml) and treatedwith N-NaOH solution (26 ml) for 4 hours by which time no dipeptideester can be detected. Most of the methanol is evaporated in vacuo andthe residue is acidified with 5 percent KHSO then extracted with ethylacetate. The organic phase is washed with water, dried over Na SO andevaporated to dryness. The residue is crystallized from ethylacetate-hexane on standing for several hours to yield 1 1.1 gr of theabove-titled product (85 percent); mp.

Anal. Cald. for C H N SO (603.6): C 59.69, H 6.85, N 6.96. Found: C59.28, H 6.80, N 7.15.

EXAMPLE 3 N-tert-Butyloxycarbonyl-L-Phenylalanyl-L- Phenylalanine MethylEster Boc-Phe-OH (53 gr, 0.2 moles) is dissolved in a mixture oftetrahydrofuran-dimethylformamide 1:1) (500 m1), mixed withN-methylmorpholine (22 m1) and cooled at 15C, then isobutylchloroformate(27 ml) is added under stirring. The reaction mixture is stirred for 7minutes and then a solution of PheOMe HCl (44 gr, 0.2 moles) andN-methylmorpholine (22 ml) in dimethylformamide is added and the mixtureis allowed to reach room temperature overnight.

The triethylamine hydrochloride salt which separates is filtered out andthe filtrate is evaporated to a small volume. The residue is treatedwith an excess of water to give an oily compound which crystallizesafter 30 minutes. The crystalline solid is washed on the filter with 5percent KHSO water, 5 percent KHCO water and dried to afford a solid ina yield of 80 gr (96 percent) of the above-titled product; R,(EtOAc-Hexane, 1:1 0.90.

Anal. Cald. for C ,H N O (426.5 C 67.59, H 7.09, N 6.56. Found: C 67.09,H 6.97, N 6.63

EXAMPLE 4 N-tert-Butyloxycarbonyl-L-Phenylalanyl-L- PhenylalanineBOC-Phe-Phe-OMe 122 gr, 0.29 moles) of Example 3 is dissolved inmethanol-dioxane-acetone (ca. 1000 ml, 1: 1:1 and treated with l Nsodium hydroxide solu- R; (CHCl tion for 3 hours. The basic solution isneutralized with dilute hydrochloric acid (pH 7) and then most of theorganic solvent is removed in vacuo. The residue is diluted with waterand acidified with 5 percent KHSO aqueous, to afford a crystalline solidwhich is washed with water and dried to yield 73 gr (62 percent) of theabove-titled product; mp. 93-95C.

Anal. Cald. for C H N O (412): C 66.99, H 6.80, N 6.80. Found: C 66.62,H 6.68, N 6.78

EXAMPLE 5 N-tert-Butyloxycarbonyl-L-Phenylalanyl-L-Phenylalanyl-L-tryptophan Methyl Ester BOC-Phe-Phe-OH (41.2 gr, 0.1moles) of Example 4 is dissolved in dimethylformamide-tetrahydrofuran(250 ml, 1:121) and mixed with N-hydroxysuccinimide (14 gr), thenTrp-OMe.HC1 (25.45 gr, 0.1 moles) and triethylamine (13.9 ml). Themixture is cooled in an ice bath and treated with DCC (21 gr, 0.12moles) for 2 hours in the ice bath then overnight at room temperature.The dicyclohexylurea (DCU) which separates is filtered off and thefiltrate is concentrated to half its volume then an excess of water isadded to afford a gummy solid which is taken in EtOAc and washed with 5percent KHSO water, 5 percent KHCO water, dried over Na SO, andevaporated to dryness. The residue is solidified from EtOAc-Et O-hexane,then from hexane, to afford a powder in a yield of 57 gr (93 percent) ofthe above-titled product. R,(EtOAc-Heptane) 0.55 and an impurity at0.05.

A portion of this material (10 gr) is chromatographed through a columnof silica gel (4.5 X 55 cm) and eluted with EtOAc-Hexane (1:1) to give awhite solid, 6.4 gr (64 percent) chromatographically homogeneous. mp.113-116C dec.

Anal. Calc. for C ,,H N O (612.7): C 68.61 H 6.58, N 9.15. Found: -C68.74, H 6.70, N 9.32.

EXAMPLE 6 N-tert-Butyloxycarbonyl-L-Phenylalanyl-L-Phenylalanyl-L-Tryptophan BOC-Phe-Phe-Trp-OMe (5.5 gr, 9 m moles) isdissolved in methanol (25 m1) and treated with 1 N-NaOH (12 ml) for 3hours at room temperature. The organic solvent is evaporated to a smallvolume and the residue is diluted with water then acidified with 5percent KHSO, to give a white precipitate which is filtered, washed withwater and dried. Yield is 5 gr, (93 percent); R, (CHCl -MfiOH-ACOH,10:5) 0.80 trace at 0.05; mp. -142C.

Anal. Cald. for C H ,,N O .3 H O (652.6): C 62.56, H 6.79, N 8.57.Found: C 62.52, H 6.03, N 8.76.

EXAMPLE 7 N-tert-Butyloxycarbonyl-L-Phenylalanyl-L-Phenylalanyl-L-Tryptophyl- N -Benzyloxycarbonyl-L-Lysine Methyl EsterBoc-Phe-Phe-Trp-OH (10 gr, 16.7 m moles) of Example 6 is mixed withLys(CBz)OMe HCl salt (5.6 gr, 17 m moles) in dimethylformamide ml) andtriethylamine (2.32 ml) is added followed by N- hydroxysuccinimide (2.3gr). The mixture is cooled in an ice bath then 4.12 gr ofdicyclohexylcarbodiimide (DCC) is added under stirring. It is kept for 2hours in the ice bath and for 20 hours at room temperature after whichtime the DCU which separates is filtered off and the filtrate is treatedwith an excess of water to precipitate a gummy material. This materialis taken in EtOAc and the organic phase is washed with percent citricacid, brine. 5 percent Na Co brine, and dried over Na SO for a shorttime then concentrated to a small 5 volume and treated with an excess ofEt O to afford a crystalline solid in a yield of 1 1.7 gr (80 percent)of the above-titled product; mp. 159 460C; R, (CHCI MeOH-AcOl-l,85:10:5) 0.75.

EXAMPLE 8 N tert-Butyloxycarbonyl-L-Phenylalanyl-L-Pheylalanyl-L-Tryptophyl- N Benzyloxycarbonyl-L-LysineBOC-Phe-Phe-Trp-Lys(Cbz)OMe (8.8 gr, 10 m moles) of Example 7 isdissolved in a mixture of methanol-acetone (150 ml, 1:1) and treatedwith 1 N- NaOH 12 ml) for 6 hours at room temperature. Acidificationwith 10 percent citric acid (200 ml) gives a solid which is filtered andwashed with water thoroughly to yield 8.5 gr (99 percent) of theabove-titled product; mp. l66168C; [a],, =8.91 (C 1, DMF); R, (CHCl-MeOH, 10:1) 0.30, R, (n-Butanol-waterpyridine-acetic acid 30:24:20:o)0.90.

Anal. Cald. for C H N (860.9): C 66.69, H 6.56, N 9.76. Found: C 66.39,N 6.63, N 9.63.

EXAMPLE 9 Nt-Butyloxycarbonyl-O-benzylL-Threonyl-L- Phenylalanine methylester A solution of Boc-L-Thr(Bzl)-OH (61.8 g, 0.2 moles) andN-methylmorpholine (22.4 ml, 0.2 moles) in tetrahydrofuran is cooled to15C. Isobutylchloroformate (26.2 ml, 0.2 moles) is added in portions,keeping the temperature between 15 and 10C. After stirring at 15C for 15minutes, a cold mixture of L-Phe-OMe HCl (43.1 g, 0.2 moles) andN-methylmorpholine (22.4 ml, 02 moles) in dimethylformamide is added inportions keeping the temperature between 10 and 5C. The mixture isstirred at 0C for 2 hours, and then at room temperature overnight. Thefiltered reaction mixture is concentrated in vacuo, and the residuetaken up in ethyl acetate. The ethyl acetate solution is washedconsecutively with 5 percent KHSO.,, 5 percent KHCO saline, and driedover Na SO After concentrating in vacuo an oil is obtained whichcrystallizes on standing. The solid is recrystallized from isopropylether-hexane to yield 74.9 g (80 percent) of the abovetitled product;mp. 78-8lC; [01) 10.75 (c 1.023, MeOl-l); R, (CHCI 0.35.

Anal. Cald. for C H N O (470.55): C 66.36, H 7.28, N 5.95. Found: C66.72, H 7.32, N 5.85.

EXAMPLE l0 N-t-Butyloxycarbonyl-O-Benzyl-L-Threonine-L- PhenylalanineBOC-Thr(Bzl)-Phe-OMe (23.5 gr, 0.05 moles) of Example 9 is dissolved ina mixture of MeOH dioxane (200 m1, 1:1) and treated with lN-NaOH (55 ml)for 3 hours (until starting material cannot be detected by TLC). Most ofthe solvent is evaporated in vacuo and the residue is diluted with waterthen it is acidified with 5 percent citric acid. The gum which separatesis taken in EtOAc and washed with water (brine) then evaporated todryness. The esidue is crystallized from Et,0- Hexane to yield 19.8 gr(87 percent) of the above-titled product; mp. 118l19C; R,(chloroform-methanolglacial acetic acid, 85: 10:5) 0.80.

EXAMPLE 1 l N-tert-Butyloxycarbonyl-ObenzylL-threonyl-O- benzyl'L-serinemethyl ester N-tert-butyloxycarbonyl-O-benzyl-L-threonine (30.9 gr, 0.1mole) is dissolved in dry tetrahydrofuran (200 ml) cooled at 20C andtreated with N- methylmorpholine (1 1 ml) followed byisobutylchloroformate 13.4 ml). The cold reaction mixture is stirred for5 minutes at -20C then treated with a solution of O-benzyl-L-serinemethyl ester hydrochloride (25 g, ca. 0.1 moles) containingN-methylmorpholine (1 1 ml), in dimethylformamide, and the mixture isallowed to reach room temperature overnight.

The solvent is removed in vacuo and the residue is partitioned betweenwater-ethyl acetate. The organic phase is washed with 5 percent citricacid, water, aq. KHCO water and dried over MgSO then evaporated todryness to afford an oily residue which crystallizes from ethylether-hexane to a jelly like solid in yield of 29 gr; R, (CHCl Meol-l,25:1) 0.85; R; (EtOAc hexane, 1:1) 0.65.

EXAMPLE l2 N-tert-Butyloxycarbonyl-O-benzyl-L-threonyl-O-benzyl-L-serine BOC-Thr(Bzl) -Ser( B21) OMe (28.2 gr, 0.0563 moles) ofExample 11 is dissolved in methanol (ca. 50 ml) and treated with 1 Nsodium hydroxide ml) for 1.5 hours at room temperature. The alkalinesolution is neutralized to pH 7 with 10 percent citric acid and most ofthe methanol is removed in vacuo. The residue is diluted with some waterand acidified with 5 percent aq. KHSO, then extracted with EtOAc. Theorganic phase is washed with H O dried over Na SO and evaporated todryness to an oil. The yield is quantitative; R, (EtOAc-hexane, 1:1)0.15 (long spot).

EXAMPLE 1 3 N-tert-Butyloxycarbonyl-O-benzyl-L-threonyl-O-benzyl-L-seryl-S-pmethoxybenzyl-L-cysteine benzyl ester BOC-Thr(Bzl)-Ser(Bzl)-OH (24.3 gr, 50 m moles) of Example 12 is dissolved inacetonitrile and dichlormethane (250 ml, 2:3) and is mixed withHCys(SMBzl)OBzl TosOH (25 gr, 50 m moles), then with triethylamine (6.9m1) and N- hydroxybenzotriazole (6.8 gr) and the mixture is cooled in anice bath.

A solution of DCC l 1 gr, 53 m moles) in acetonitrile (50 ml) is addedand the reaction mixture is stirred for 2 hours in the cold then for 2days at room temperature. The DCU which separates is filtered off andthe filtrate is evaporated to dryness. The oil residue is partitionedbetween ethyl acetate-water and the organic phase is washed with 10percent citric acid, water, KHCO brine and dried over Na SO The solventis evaporated and the oily residue is crystallized from Et- O-hexane toafford a white solid in a yield of 24.5 gr of the above-titled product;mp. 87-90C; [01],, l4.7 (C 0.98, DMF); R,(chloroform-methanol, 25:1)0.54, traces at 0.4 and 0.3, (heptane-EtOAc, 1:1 0.64, traces at 0.25; 1positive.

Anal. Calc. for C H N SO (798.9): C 66.15, H 6.56, N 5.26. Found: C66.22, H 6.95, N 5.29.

EXAMPLE [4 O-Benzyl-L-Threonyl-O-Benzy1-L-Sery1-S-pMethoxybenzyl-L-Cysteine Benzyl Ester, TrifluoroacetateBOC-Thr(Bzl)-Ser(Bzl)-Cys(SMBzl)-OBzl (8 gr, 10 m moles) of Example 13is mixed with anisole (100 m moles) and treated with trifiuoroaceticacid (100 ml) for 45 minutes. The solvent is evaporated in vacuo and theresidue is dissolved in dry Et O, then evaporated to dryness in highvacuo to give an oily compound in a yield of 7.2 gr (90 percent) of theabove-titled product. R, (BWA, 4: 1:1) 0.9; R; (Heptane-EtOAc) 0.0-0.1long spot, 1 positive and ninhydrin positive.

EXAMPLE l5 N-tert-Butyloxycarbonyl-O-benzyl-L-Threonyl-L-Phenylalanyl-O-Benzyl- L-Threonyl-O-Benzyl-L-Seryl-S-p-methoxyBenzyl-L-Cysteine Benzyl Ester BOC-Thr( B21 )-Phe-OH (9.2 gr, 20 mmoles) of Example 10 is dissolved in dimethylformamide (100 ml) andmixed with N-hydroxysuccinimide (3.4 gr) and a solution of Thr(Bzl)-Ser(Bzl)-Cys (SMBzl)OBzl TFA salt (20 m moles) of Example 14 indimethylformamide (20 ml) is neutralized with triethylamine to pH 7. Themixture is cooled in an ice bath and treated with DCC (4.5 gr) for 2hours in the cold then for 2 days at room temperature. The DCU isfiltered and the filtrate evaporated to dryness. The residue istriturated with water to give a precipitate which is taken in EtOAc,washed with 5 percent citric acid, water 5 percent Na CO water driedover Na SO and evaporated to dryness. The residue is solidified from EtO-HeXane to yield 17.3 gr (70 percent) of the above-titled product; mp.105-107C; 3.6 (c l, DMF); R; (CHCI MeOH, 10:1) 0.90.

Anal. Cald. for C H N SO H O (1156.2): C 66.46, H 6.71, N 6.05. Found: C66.68, H 6.59, N 6.20.

EXAMPLE l6 N-tert-Butyloxycarbonyl-L-Phenylalanyl-L-Phenylalanyl-L-Tryptophyl- N -Benzyloxycarbonyl-L-Lysyl-O-Benzyl-L-Threonyl-L-Phenylalanyl- O-Benzyl-L-Threonyl-O-Benzyl-L-Seryl-S-p-Methoxybenzyl-L-Cysteine Benzyl Ester BOC-Thr( Bzl)-Phe-Thr( Bzl )-Ser(Bzl)-Cys( SMBzl OBzl (11.4 gr, 10 m moles) of Example is mixed with 200m moles anisole ml) and treated with trifluoroacetic acid (200 ml) for30 minutes in an ice bath and for 30 minutes further at roomtemperature, then the mixture is evaporated to dryness in high vacuo andthe residue triturated with an excess of dry Et O- pentane to afford asolid in a yield of l 1 gr (95 percent) of the above-titled product; R,(CHCl -MeOH-AcOH, 85:l0:5) 0.55 trace at 0.45.

The above solid (9.37 gr, 8.14 m moles) is dissolved in DMF (ca. 150 ml)and neutralized with triethylamine (1.14 ml) then mixed withBOC-PhePhe-Trp-Lys- (Cbz)-OH (7 gr, 8.14 m moles) of Example 12 and N-hydroxysuccinimide (1.035 gr) and the mixture is cooled in an ice bath.To the cold mixture DCC (2.06 gr) is added under stirring for 2 hours inthe ice bath and for 3 days at room temperature. The DCU which separatesis filtered off and the filtrate is treated with an excess of water togive a solid which is washed on the filter thoroughly with 5 percentcitric acid, water, 5 percent Na CO water and dried to yield 18 gr (96percent) of the above-titled product mp. 200203C dec; [011 5.6 (C 0.5,DMF); R, (CHCl -MeOH, 10:1) 0.65 R (CHCI -MeOH-AcOH, :10:5) 0.83.

Anal. Cald. for C H N SO H O (1181.04): C 67.60, H 6.52, N 8.10. Found:C 67.31, H 6.60, N 8.46.

EXAMPLE 17 L-Phenylalanyl-L-PhenylalanyL-L-Tryptophyl-N-Benzyloxycarbonyl-L- Lysyl-O-Benzyl-L-Threonyl-L-Phenylalanyl-O-BenzylL-Threonyl-O- Benzyl L-Seryl-s-p-Methoxybenzyl-L-Cysteine Benzyl EsterTrifluoroacetate BOC-Phe-Phe-Trp-Lys( Cbz )-Thr( Bzl )-Phe-Thr(Bzl)-Ser( Bzl)-Cys (SMBZUOBZI (3 gr, 1.6 m moles) of Example 16 ismixed with anisole (2 ml, ca. 32 m moles) and then an excess oftrifluoroacetic acid is added. The solution is left to stand at roomtemperature for 1 hour then evaporated to dryness and the residuetriturated with dry Et O to afford a solid in a yield of 2.8 gr (92percent) of the above-titled product; R, (chloroform-methanol-aceticacid, 85:10:5) 0.50; R, (chloroform-methanol, 10:1) 0.55, trace at 0.25;ninhydrin and 1 positive spots. Amino Acid analysis*: Thr (2) 1.97; Ser(1) 0.76; Phe (3) 3.00; Lys (1) 0.90.

* Numbers in brackets indicate theoretical quantity of the amino acids.

EXAMPLE 1 8 N-tert-butyloxycarbonyl-S-pmethoxybenzyl-L- cysteinyl-N-benzyloxy carbonyl-L-lysyl-L-phenylalanyl-L-phenylalanyl-L-tryptophyl-N benzyloxycarbonyl-L-lysyl-O-benzyl-L-threonyl-Lphenylalanyl-O-benzyl-L-threonylO- benzylL-seryl-S-p-methoxybenzylL-cysteine benzyl ester H-Phe-Phe-Trp-Lys( Cbz )-Thr( Bzl )-Phe-Thr( BzlSer( Bzl)-Cys (SMBzl)-Obzl.TFA salt (1 gr, 0.53 m moles) of Example 17is dissolved in DMF 15 ml) and neutralized with triethylamine (0.072 ml)then mixed with N-hydroxysuccinimide (60 mgr) followed by BOC-Cys(SMBzl)-Lys(Cbz)-OH (320 mgr, 0.53 m moles) obtained in Example 2. Themixture is cooled in an ice bath and treated with DCC mgr) for 2 hoursin the cold and for 3 days at room temperature.

The DCU which separates is filtered off and the filtrate is treated withan excess of brine to give a white solid which is washed with water,abs. EtOH, MeOH and finally with Et O to yield 1.04 gr (83 percent) ofthe above titled product; mp. 225-235C dec. This product isrecrystallized from DMF-H O.

Anal. Calcd. for c u N s o (2366.5): C 66.99, H 6.47, N 8.28, S 2.70.Found: C 67.10, H 6.54, N 8.36, S 3.38

EXAMPLE l9 L-cystinyl-L-lysyl-L-phenylalanyl-L-phenylalanyl-L-tryptophyl-L- lysyl-L-threonyl-L-phenylalanyl-L-threonyl-L-seryl-L-cystine (cyclic 1, l1 disulfide) triacetate salt.

Lys( Cbz )-Thr( Bzl )-Phe-Thr( Bzl )-Ser( B21 Cys(SMBal)-OBzl (1 gr,0.42 m moles) of Example 18 is mixed with 4 ml anisole and treated for30 minutes at room temperature with liquid HF after which time theexcess HF is evaporated as fast as possible (ca. 1 hour) to yield thedeprotected compound L-cysteinyl-L-lysyl-L-phenylalanyl-L-phenylalanyl-L-tryptophyl-Llysyl-L-threonyl-L-phenylalanyl-L-threonyl-L-seryl-L- cysteine. Thisresidue is washed with dry Et O (flushed with hydrogen) and then in 0.01M ammonium acetate solution (2 l) which is flushed with nitrogen. The pHis brought to 9.2 with a few drops of dilute NH OH and left to stand for2 days, then lyophilized twice to yield a white fluffy solid 61 1 mgr100 percent, some NH OCOCH present).

This crude product (556 mgr) is chromatographed through a column ofSephadex (3-25 (2.5 X 30 cm) which is equilibrated first with the lowerphase of a biphasic system, n-butanol-acetic acid-water, 4:115, thenwith the upper phase of the system. Fractions of 6.9 ml volume arecollected and the material is located by the Folin-Lowry method.

Three main peaks emerge from the column between fractions 53-72 (A),73-79 (B) and 8095 (C). The fractions 80-95 (C) are pooled andlyophilized to yield 84 mgr of a fluffy solid which is the above titledproduct.

R, (n-butanoLwater-acetic acid) 0.45; R, (isopropanol-l N-NH OH. 2: l)0.75.

Amino acid analysis: Thr (2) 1.81; Ser (1) 0.93; Phe (3) 2.97; Lys (2)2; Cys (2) 1.70; Trp (1) not determined.

The growth hormone release inhibiting activity of the compound ofExample 19 was determined by radioim- Lu) carboxyl activation (2)coupling munoassay in a rat pituitary cell culture system as describedby Vale et a1.. Endocrinology 91, pp 562 (1972) and Grant et al.,Biochemical and Biophysical Research Communications 51, pp 100-106(1973). The compound of Example 19 was found active in inhibiting growthhormone release at a concentration as low as ng/ml.

The compound of formula I described herein may be administered to warmblooded mammals, including humans, either intravenously, subcutaneously,intramuscularly or orally to inhibit the release of growth hormone wherethe host being treated requires therapeutic treatment for excesssecretion of somatotropin which is associated with conditions such asjuvenile diabetes and acromegaly. The contemplated dose range for oraladministration in tablet or capsule form to large mammals is about 0.015mg to about 7 mg/kg of body weight per day while the dose range forintravenous injection in an aqueous solution is about 0.1 g to about0.15 mg/kg of body weight per day. When administered subcutaneously orintramuscularly a dose range of about 1.5 g to about 7 mg/kg of bodyweight per day is contemplated. Obviously, the required dosage will varywith the particular condition being treated, the severity of thecondition and the duration of treatment.

If the active ingredient is administered in tablet form the tablet maycontain: a binder such as gum tragacanth, corn starch, gelatin, anexcipient such as dicalcium phosphate; a disintegrating agent such ascorn starch, alginic acid, etc.; a lubricant such as magnesium stearate;and a sweetening and/or flavoring agent such as sucrose, lactose,Wintergreen, etc. Suitable liquid carriers for intravenousadministration include isotonic saline, phosphate buffer solutions, etc.

FLOW DIAGRAM Thr (R -Ser (R Cys (R OR What is claimed is: 1. Anundecapeptide selected from those of the formula:

H-6ys-Lys-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Serys- OH,

HCys-Lys-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-CysOH and the non-toxic acidaddition salts thereof, said amino acid residues in said undecapeptidehaving an asymmetric a-carbon being of the L-configurationi 2. Acompound according to claim 1 which is: L-cysteinyl-L-lysyl-L-phenylalanyl-L-phenylalanyl-L-tryptophyl-L-lysyl-L-threonylL-phenylalanyl-L-threonyl-L-seryLL-cysteine and a non-toxic acid addition salt thereof.

3. An undecapeptide of the formula:

R is selected from the group consisting of hydrogen and a protectinggroup for the side chain amino substituent of the lysine residueselected from the group consisting of benzyl, chlorobenzyloxycarbonyl,tosyl, 2,4-dinitrophenyl, t-amyloxycarbonyl, t-butyloxycarbonyl andbenzyloxycarbonyl;

R and R are protecting groups for the alcoholic hydroxyl group of thethreonine and serine residues selected from the group consisting ofacetyl, tosyl, benzoyl, tert-butyl, trityl, benzyl andbenzyloxycarbonyl; and

R is a a-carboxyl protecting group selected from the class consisting ofC -C alkyl, benzyl, substituted benzyl, phenacyl, phthalimidomethyl,3methylthioethyl, 4-picolyl and 4-(methylthio) phenyl, said substituenton benzyl being selected from at least one of methyl, methoxy and nitro;and the acid addition salts thereof, said amino acid residues in saidundecapeptide having an asymmetric a-carbon atom being of theL-configuration.

4. A compound according to claim 3 wherein R is tert-butyloxycarbonyl.

5. A compound according to claim 4 wherein R is p methoxybenzyl, R isbenzyloxycarbonyl, R and R are benzyl and R is benzyl.

6. A compound according to claim I which is: L-cystinyl-L-lysyl-L-phenylalanyl-L-phenylalanyl-L-tryptophyl-L-lysyl-L-treonyl-L-phenylalanyl-L-threonyl-L-seryl-L-cystine (cyclic 1, ll disulfide) and a non-toxic acidaddition salt thereof.

1. An undecapeptide selected from those of the formula:H-Cys-Lys-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys-OH,H-Cys-Lys-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys-OH and the non-toxic acidaddition salts thereof, said amino acid residues in said undecapeptidehaving an asymmetric Alpha -carbon being of the L-configuration.
 2. Acompound according to claim 1 which is:L-cysteinyl-L-lysyl-L-phenylalanyl-L-phenylalanyl-L-tryptophyl-L-lysyl-L-threonyl-L-phenylalanyl-L-threonyl-L-seryl-L-cysteine and a non-toxicacid addition salt thereof.
 3. AN UNDECAPEPTIDE OF THE FORMULA:
 4. Acompound according to claim 3 wherein R is tert-butyloxycarbonyl.
 5. Acompound according to claim 4 wherein R1 is p-methoxybenzyl, R2 isbenzyloxycarbonyl, R3 and R4 are benzyl and R5 is benzyl.
 6. A compoundaccording to claim 1 which is:L-cystinyl-L-lysyl-L-phenylalanyl-L-phenylalanyl-L-tryptophyl-L-lysyl-L-treonyl-L-phenylalanyl-L-threonyl-L-seryl-L-cystine (cyclic 1, 11disulfide) and a non-toxic acid addition salt thereof.